During aging, organisms show altered gene expression patterns and have an increasingly impaired ability to respond to stress-causing and mitogenic stimuli. Since post-transcriptional processes critically regulate changes in the collections of expressed proteins, it is extremely important to elucidate the microRNAs (as well as RBPs, as described in other projects) that control age-related gene expression patterns. To investigate microRNA function during senescence, we employ approaches such as microRNA reduction (by transfecting an antisense RNA), microRNA overexpression (by transfecting a precursor of the microRNA), and the identification of microRNA-associated mRNAs by transfecting biotinylated microRNAs and identifying target mRNAs through various methods (eg, microarray, RT-PCR). We investigate whether microRNAs affect the stability of target mRNAs during senescence by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of microRNA abundance. We investigate whether microRNAs affect the translation of target mRNAs by modulating microRNA levels, and subsequently studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by microRNAs and use different senescence-associated markers to examine changes in the senescence phenotype. During the past funding period, we have shown that the translation of MKK4 (a protein upregulated during replicative senescence) was repressed by miR-15b, miR-24, miR-25, and miR-141. These four microRNAs are downregulated with senescence, as were other microRNAs identified by microRNA microarray analysis. These studies also identified other microRNAs whose levels were significantly higher or lower in senescent cells than in early-passage, young cells. We also identified miR-519 as a key microRNA implicated in downregulating HuR expression. Ongoing studies are analyzing systematically the target mRNAs of biotinylated microRNAs whose levels decline or increase with senescence; target mRNAs are then validated using microarrays.